Method for the production of glycerol by recombinant organisms

ABSTRACT

Recombinant organisms are provided comprising genes encoding a glycerol-3-phosphate dehydrogenase and/or a glycerol-3-phosphatase activity useful for the production of glycerol from a variety of carbon substrates. The organisms further contain disruptions in the endogenous genes encoding proteins having glycerol kinase and glycerol dehydrogenase activities.

FIELD OF INVENTION

This application is a divisional of U.S. application Ser. No.09/695,786, which is a divisional of U.S. application Ser. No.08/982,783 filed 3 Dec. 1997 (now abandoned), which is acontinuation-in-part of U.S. application Ser. No. 08/968,418 filed 12Nov. 1997, (now abandoned), claiming benefit to U.S. ProvisionalApplication No. 60/030,602, filed 13 Nov. 1996.

The present invention relates to the field of molecular biology and theuse of recombinant organisms for the production of glycerol andcompounds derived from the glycerol biosynthetic pathway. Morespecifically the invention describes the construction of a recombinantcell for the production of glycerol and derived compounds from a carbonsubstrate, the cell containing foreign genes encoding proteins havingglycerol-3-phosphate dehydrogenase (G3PDH) and glycerol-3-phosphatase(G3P phosphatase) activities where the endogenous genes encoding theglycerol-converting glycerol kinase and glycerol dehydrogenaseactivities have been deleted.

BACKGROUND

Glycerol is a compound in great demand by industry for use in cosmetics,liquid soaps, food, pharmaceuticals, lubricants, anti-freeze solutions,and in numerous other applications. The esters of glycerol are importantin the fat and oil industry. Historically, glycerol has been isolatedfrom animal fat and similar sources; however, the process is laboriousand inefficient. Microbial production of glycerol is preferred.

Not all organisms have a natural capacity to synthesize glycerol.However, the biological production of glycerol is known for some speciesof bacteria, algae, and yeast. The bacteria Bacillus licheniformis andLactobacillus lycopersica synthesize glycerol. Glycerol production isfound in the halotolerant algae Dunaliella sp. and Asteromonas gracilisfor protection against high external salt concentrations (Ben-Amotz etal., (1982) Experientia 38:49-52). Similarly, various osmotolerant yeastsynthesize glycerol as a protective measure. Most strains ofSaccharomyces produce some glycerol during alcoholic fermentation andthis production can be increased by the application of osmotic stress(Albertyn et al., (1994) Mol. Cell. Biol. 14, 4135-4144). Earlier thiscentury glycerol was produced commercially with Saccharomyces culturesto which steering reagents were added such as sulfites or alkalis.Through the formation of an inactive complex, the steering agents blockor inhibit the conversion of acetaldehyde to ethanol; thus, excessreducing equivalents (NADH) are available to or “steered” towardsdihydroxyacetone phosphate (DHAP) for reduction to produce glycerol.This method is limited by the partial inhibition of yeast growth that isdue to the sulfites. This limitation can be partially overcome by theuse of alkalis which create excess NADH equivalents by a differentmechanism. In this practice, the alkalis initiated a Cannizarrodisproportionation to yield ethanol and acetic acid from two equivalentsof acetaldehyde. Thus, although production of glycerol is possible fromnaturally occurring organisms, production is often subject to the needto control osmotic stress of the cultures and the production ofsulfites. A method free from these limitations is desirable. Productionof glycerol from recombinant organisms containing foreign genes encodingkey steps in the glycerol biosynthetic pathway is one possible route tosuch a method.

A number of the genes involved in the glycerol biosynthetic pathway havebeen isolated. For example, the gene encoding glycerol-3-phosphatedehydrogenase (DAR1, GPD1) has been cloned and sequenced fromSaccharomyces diastaticus (Wang et al., (1994), J. Bact. 176:7091-7095).The DAR1 gene was cloned into a shuttle vector and used to transform E.coli where expression produced active enzyme. Wang et al., supra,recognizes that DAR1 is regulated by the cellular osmotic environmentbut does not suggest how the gene might be used to enhance glycerolproduction in a recombinant organism.

Other glycerol-3-phosphate dehydrogenase enzymes have been isolated. Forexample, sn-glycerol-3-phosphate dehydrogenase has been cloned andsequenced from S. cerevisiae (Larason et al., (1993) Mol. Microbiol.,10:1101). Albertyn et al., (1994) Mol. Cell. Biol., 14:4135) teach thecloning of GPD1 encoding a glycerol-3-phosphate dehydrogenase from S.cerevisiae. Like Wang et al., both Albertyn et al. and Larason et al.recognize the osmo-sensitvity of the regulation of this gene but do notsuggest how the gene might be used in the production of glycerol in arecombinant organism.

As with G3PDH, glycerol-3-phosphatase has been isolated fromSaccharomyces cerevisiae and the protein identified as being encoded bythe GPP1 and GPP2 genes (Norbeck et al., (1996) J. Biol. Chem.,271:13875). Like the genes encoding G3PDH, it appears that GPP2 isosmotically-induced.

Although the genes encoding G3PDH and G3P phosphatase have beenisolated, there is no teaching in the art that demonstrates glycerolproduction from recombinant organisms with G3PDH/G3P phosphataseexpressed together or separately. Further, there is no teaching tosuggest that efficient glycerol production from any wild-type organismis possible using these two enzyme activities that does not requireapplying some stress (salt or an osmolyte) to the cell. In fact, the artsuggests that G3PDH activities may not affect glycerol production. Forexample, Eustace ((1987), Can. J. Microbiol., 33:112-117)) teacheshybridized yeast strains that produced glycerol at greater levels thanthe parent strains. However, Eustace also demonstrates that G3PDHactivity remained constant or slightly lower in the hybridized strainsas opposed to the wild type.

Glycerol is an industrially useful material. However, other compoundsmay be derived from the glycerol biosynthetic pathway that also havecommercial significance. For example, glycerol-producing organisms maybe engineered to produce 1,3-propanediol (U.S. Pat. No. 5,686,276), amonomer having potential utility in the production of polyester fibersand the manufacture of polyurethanes and cyclic compounds. It is knownfor example that in some organisms, glycerol is converted to3-hydroxypropionaldehyde and then to 1,3-propanediol through the actionsof a dehydratase enzyme and an oxidoreductase enzyme, respectively.Bacterial strains able to produce 1,3-propanediol have been found, forexample, in the groups Citrobacter, Clostridium, Enterobacter,Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. Glyceroldehydratase and diol dehydratase systems are described by Seyfried etal. (1996) J. Bacteriol. 178:5793-5796 and Tobimatsu et al. (1995) J.Biol. Chem. 270:7142-7148, respectively. Recombinant organisms,containing exogenous dehydratase enzyme, that are able to produce1,3-propanediol have been described (U.S. Pat. No. 5,686,276). Althoughthese organisms produce 1,3-propanediol, it is clear that they wouldbenefit from a system that would minimize glycerol conversion.

There are a number of advantages in engineering a glycerol-producingorganism for the production of 1,3-propanediol where conversion ofglycerol is minimized. A microorganism capable of efficiently producingglycerol under physiological conditions is industrially desirable,especially when the glycerol itself will be used as a substrate in vivoas part of a more complex catabolic or biosynthetic pathway that couldbe perturbed by osmotic stress or the addition of steering agents (e.g.,the production of 1,3-propanediol). Some attempts at creating glycerolkinase and glycerol dehydrogenase mutants have been made. For example,De Koning et al. (1990) Appl. Microbiol Biotechnol. 32:693-698 reportthe methanol-dependent production of dihydroxyacetone and glycerol bymutants of the methylotrophic yeast Hansenula polymorpha blocked indihydroxyacetone kinase and glycerol kinase. Methanol and an additionalsubstrate, required to replenish the xyulose-5-phosphate co-substrate ofthe assimilation reaction, were used to produce glycerol; however, adihydroxyacetone reductase (glycerol dehydrogenase) is also required.Similarly, Shaw and Cameron, Book of Abstracts, 211th ACS NationalMeeting, New Orleans, La., Mar. 24-28 (1996), BIOT-154 Publisher:American Chemical Society, Washington, D.C., investigate the deletion ofof ldhA (lactate dehydrogenase), glpK (glycerol kinase), and tpiA(triosephosphate isomerase) for the optimization of 1,3-propanediolproduction. They do not suggest the expression of cloned genes for G3PDHor G3P phosphatase for the production of glycerol or 1,3-propanediol andthey do not discuss the impact of glycerol dehydrogenase.

The problem to be solved, therefore, is the lack of a process to directcarbon flux towards glycerol production by the addition or enhancementof certain enzyme activities, especially G3PDH and G3P phosphatase whichrespectively catalyze the conversion of dihydroxyacetone phosphate(DHAP) to glycerol-3-phosphate (G3P) and then to glycerol. The problemis complicated by the need to control the carbon flux away from glycerolby deletion or decrease of certain enzyme activities, especiallyglycerol kinase and glycerol dehydrogenase which respectively catalyzethe conversion of glycerol plus ATP to G3P and glycerol todihydroxyacetone (or glyceraldehyde).

SUMMARY OF THE INVENTION

The present invention provides a method for the production of glycerolfrom a recombinant organism comprising: transforming a suitable hostcell with an expression cassette comprising either one or both of (a) agene encoding a protein having glycerol-3-phosphate dehydrogenaseactivity and (b) a gene encoding a protein having glycerol-3-phosphatephosphatase activity, where the suitable host cell contains a disruptionin either one or both of (a) a gene encoding an endogenous glycerolkinase and (b) a gene encoding an endogenous glycerol dehydrogenase,wherein the disruption prevents the expression of active gene product;culturing the transformed host cell in the presence of at least onecarbon source selected from the group consisting of monosaccharides,oligosaccharides, polysaccharides, and single-carbon substrates, wherebyglycerol is produced; and recovering the glycerol produced.

The present invention further provides a process for the production of1,3-propanediol from a recombinant organism comprising: transforming asuitable host cell with an expression cassette comprising either one orboth of (a) a gene encoding a protein having glycerol-3-phosphatedehydrogenase activity and (b) a gene encoding a protein havingglycerol-3-phosphate phosphatase activity, the suitable host cell havingat least one gene encoding a protein having a dehydratase activity andhaving a disruption in either one or both of (a) a gene encoding anendogenous glycerol kinase and (b) a gene encoding an endogenousglycerol dehydrogenase, wherein the disruption in the genes of (a) or(b) prevents the expression of active gene product; culturing thetransformed host cell in the presence of at least one carbon sourceselected from the group consisting of monosaccharides, oligosaccharides,polysaccharides, and single-carbon substrates whereby 1,3-propanediol isproduced; and recovering the 1,3-propanediol produced.

Additionally, the invention provides for a process for the production of1,3-propanediol from a recombinant organism where multiple copies ofendogeneous genes are introduced.

Further embodiments of the invention include host cells transformed withheterologous genes for the glycerol pathway as well as host cells whichcontain endogeneous genes for the glycerol pathway.

Additionally, the invention provides recombinant cells suitable for theproduction either glycerol or 1,3-propanediol, the host cells havinggenes expressing either one or both of a glycerol-3-phosphatedehydrogenase activity and a glycerol-3-phosphate phosphatase activitywherein the cell also has disruptions in either one or both of a geneencoding an endogenous glycerol kinase and a gene encoding an endogenousglycerol dehydrogenase, wherein the disruption in the genes prevents theexpression of active gene product.

BRIEF DESCRIPTION OF THE FIGURES, BIOLOGICAL DEPOSITS AND SEQUENCELISTING

FIG. 1 illustrates the representative enzymatic pathways involvingglycerol metabolism.

Applicants have made the following biological deposits under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for the Purposes of Patent Procedure: DepositorIdentification Int'l. Depository Reference Designation Date of DepositEscherichia coli pAH21/DH5α ATCC 98187 26 Sep. 1996 (containing the GPP2gene) Escherichia coli (pDAR1A/AA200) ATCC 98248 6 Nov. 1996 (containingthe DAR1 gene) FM5 Escherichia coli RJF10m ATCC 98597 25 Nov. 1997(containing a glpK disruption) FM5 Escherichia coli MSP33.6 ATCC 9859825 Nov. 1997 (containing a gldA disruption)

“ATCC” refers to the American Type Culture Collection internationaldepository located at 10801 University Blvd, Manassas, Va. 20110-2209U.S.A. The designation is the accession number of the depositedmaterial.

Applicants have provided 43 sequences in conformity with the Rules forthe Standard Representation of Nucleotide and Amino Acid Sequences inPatent Applications (Annexes I and II to the Decision of the Presidentof the EPO, published in Supplement No. 2 to OJ EPO, 12/1992) and with37 C.F.R. 1.821-1.825 and Appendices A and B (Requirements forApplication Disclosures Containing Nucleotides and/or Amino AcidSequences).

DETAILED DESCRIPTION OF THE INVENTION

The present invention solves the problem stated above by providing amethod for the biological production of glycerol from a fermentablecarbon source in a recombinant organism. The method provides a rapid,inexpensive and environmentally-responsible source of glycerol useful inthe cosmetics and pharmaceutical industries. The method uses amicroorganism containing cloned homologous or heterologous genesencoding glycerol-3-phosphate dehydrogenase (G3PDH) and/orglycerol-3-phosphatase (G3P phosphatase). These genes are expressed in arecombinant host having disruptions in genes encoding endogenousglycerol kinase and/or glycerol dehydrogenase enzymes. The method isuseful for the production of glycerol, as well as any end products forwhich glycerol is an intermediate. The recombinant microorganism iscontacted with a carbon source and cultured and then glycerol or any endproducts derived therefrom are isolated from the conditioned media. Thegenes may be incorporated into the host microorganism separately ortogether for the production of glycerol.

Applicants' process has not previously been described for a recombinantorganism and required the isolation of genes encoding the two enzymesand their subsequent expression in a host cell having disruptions in theendogenous kinase and dehydrogenase genes. It will be appreciated bythose familiar with this art that Applicants' process may be generallyapplied to the production compounds where glycerol is a keyintermediate, e.g., 1,3-propanediol.

As used herein the following terms may be used for interpretation of theclaims and specification.

The terms “glycerol-3-phosphate dehydrogenase” and “G3PDH” refer to apolypeptide responsible for an enzyme activity that catalyzes theconversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate(G3P). In vivo G3PDH may be NADH; NADPH; or FAD-dependent. TheNADH-dependent enzyme (EC 1.1.1.8) is encoded, for example, by severalgenes including GPD1 (GenBank Z74071×2), or GPD2 (GenBank Z35169x1), orGPD3 (GenBank G984182), or DAR1 (GenBank Z74071x2). The NADPH-dependentenzyme (EC 1.1.1.94) is encoded by gpsA (GenBank U321643, (cds197911-196892) G466746 and L45246). The FAD-dependent enzyme (EC1.1.99.5) is encoded by GUT2 (GenBank Z47047x23), or glpD (GenBankG147838), or glpABC (GenBank M20938).

The terms “glycerol-3-phosphatase”, “sn-glycerol-3-phosphatase”, or“d,l-glycerol phosphatase”, and “G3P phosphatase” refer to a polypeptideresponsible for an enzyme activity that catalyzes the conversion ofglycerol-3-phosphate and water to glycerol and inorganic phosphate. G3Pphosphatase is encoded, for example, by GPP1 (GenBank Z47047×125), orGPP2 (GenBank U18813x11).

The term “glycerol kinase” refers to a polypeptide responsible for anenzyme activity that catalyzes the conversion of glycerol and ATP toglycerol-3-phosphate and ADP. The high energy phosphate donor ATP may bereplaced by physiological substitutes (e.g. phosphoenolpyruvate).Glycerol kinase is encoded, for example, by GUT1 (GenBank U11583×19) andglpK (GenBank L19201).

The term “glycerol dehydrogenase” refers to a polypeptide responsiblefor an enzyme activity that catalyzes the conversion of glycerol todihydroxyacetone (E.C. 1.1.1.6) or glycerol to glyceraldehyde (E.C.1.1.1.72). A polypeptide responsible for an enzyme activity thatcatalyzes the conversion of glycerol to dihydroxyacetone is alsoreferred to as a “dihydroxyacetone reductase”. Glycerol dehydrogenasemay be dependent upon NADH (E.C. 1.1.1.6), NADPH (E.C. 1.1.1.12), orother cofactors (e.g., E.C. 1.1.99.22). A NADH-dependent glyceroldehydrogenase is encoded, for example, by gldA (GenBank U00006).

The term “dehydratase enzyme” will refer to any enzyme that is capableof isomerizing or converting a glycerol molecule to the product3-hydroxypropion-aldehyde. For the purposes of the present invention thedehydratase enzymes include a glycerol dehydratase (E.C. 4.2.1.30) and adiol dehydratase (E.C. 4.2.1.28) having preferred substrates of glyceroland 1,2-propanediol, respectively. In Citrobacter freundii, for example,glycerol dehydratase is encoded by three polypeptides whose genesequences are represented by dhaB, dhaC and dhaE (GenBank U09771: basepairs 8556-10223, 10235-10819, and 10822-11250, respectively). InKlebsiella oxytoca, for example, diol dehydratase is encoded by threepolypeptides whose gene sequences are represented by pddA, pddB, andpddC (GenBank D45071: base pairs 121-1785, 1796-2470, and 2485-3006,respectively).

The terms “GPD1”, “DAR1”, “OSG1”, “D2830”, and “YDL022W” will be usedinterchangeably and refer to a gene that encodes a cytosolicglycerol-3-phosphate dehydrogenase and is characterized by the basesequence given as SEQ ID NO: 1.

The term “GPD2” refers to a gene that encodes a cytosolicglycerol-3-phosphate dehydrogenase and is characterized by the basesequence given in SEQ ID NO: 2.

The terms “GUT2” and “YIL155C” are used interchangeably and refer to agene that encodes a mitochondrial glycerol-3-phosphate dehydrogenase andis characterized by the base sequence given in SEQ ID NO: 3.

The terms “GPP1”, “RHR2” and “YIL053W” are used interchangeably andrefer to a gene that encodes a cytosolic glycerol-3-phosphatase and ischaracterized by the base sequence given in SEQ ID NO: 4.

The terms “GPP2”, “HOR2” and “YER062C” are used interchangeably andrefer to a gene that encodes a cytosolic glycerol-3-phosphatase and ischaracterized by the base sequence given as SEQ ID NO: 5.

The term “GUT1” refers to a gene that encodes a cytosolic glycerolkinase and is characterized by the base sequence given as SEQ ID NO: 6.The term “glpK” refers to another gene that encodes a glycerol kinaseand is characterized by the base sequence given in GeneBank L19201, basepairs 77347-78855.

The term “gldA” refers to a gene that encodes a glycerol dehydrogenaseand is characterized by the base sequence given in GeneBank U00006, basepairs 31744316. The term “dhaD” refers to another gene that encodes aglycerol dehydrogenase and is characterized by the base sequence givenin GeneBank U09771, base pairs 2557-3654.

As used herein, the terms “function” and “enzyme function” refer to thecatalytic activity of an enzyme in altering the energy required toperform a specific chemical reaction. Such an activity may apply to areaction in equilibrium where the production of both product andsubstrate may be accomplished under suitable conditions.

The terms “polypeptide” and “protein” are used interchangeably.

The terms “carbon substrate” and “carbon source” refer to a carbonsource capable of being metabolized by host organisms of the presentinvention and particularly mean carbon sources selected from the groupconsisting of monosaccharides, oligosaccharides, polysaccharides, andone-carbon substrates or mixtures thereof.

“Conversion” refers to the metabolic processes of an organism or cellthat by means of a chemical reaction degrades or alters the complexityof a chemical compound or substrate.

The terms “host cell” and “host organism” refer to a microorganismcapable of receiving foreign or heterologous genes and additional copiesof endogeneous genes and expressing those genes to produce an activegene product.

The terms “production cell” and “production organism” refer to a cellengineered for the production of glycerol or compounds that may bederived from the glycerol biosynthetic pathway. The production cell willbe recombinant and contain either one or both of a gene that encodes aprotein having a glycerol-3-phosphate dehydrogenase activity and a geneencoding a protein having

glycerol-3-phosphatase activity. In a

on to the G3PDH and G3P phosphatase genes, the host cell will containdisruptions in one or both of a gene encoding an endogenous glycerolkinase and a gene encoding an endogenous glycerol dehydrogenase. Wherethe production cell is designed to produce 1,3-propanediol, it willadditionally contain a gene encoding a protein having a dehydrataseactivity.

The terms “foreign gene”, “foreign DNA”, “heterologous gene”, and“heterologous DNA” all refer to genetic material native to one organismthat has been placed within a different host organism.

The term “endogenous” as used herein with reference to genes orpolypeptides expressed by genes, refers to genes or polypeptides thatare native to a production cell and are not derived from anotherorganism. Thus an “endogenous glycerol kinase” and an “endogenousglycerol dehydrogenase” are terms referring to polypeptides encoded bygenes native to the production cell.

The terms “recombinant organism” and “transformed host” refer to anyorganism transformed with heterologous or foreign genes. The recombinantorganisms of the present invention express foreign genes encoding G3PDHand G3P phosphatase for the production of glycerol from suitable carbonsubstrates. Additionally, the terms “recombinant organism” and“transformed host” refer to any organism transformed with endogenous (orhomologous) genes so as to increase the copy number of the genes.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-coding) andfollowing <(3′ non-coding) the coding region. The terms “native” and“wild-type” gene refer to the gene as found in nature with its ownregulatory sequences.

The terms “encoding” and “coding” refer to the process by which a gene,through the mechanisms of transcription and translation, produces anamino acid sequence. The process of encoding a specific amino acidsequence is meant to include DNA sequences that may involve base changesthat do not cause a change in the encoded amino acid, or which involvebase changes which may alter one or more amino acids, but do not affectthe functional properties of the protein encoded by the DNA sequence.Therefore, the invention encompasses more than the specific exemplarysequences. Modifications to the sequence, such as deletions, insertions,or substitutions in the sequence which produce silent changes that donot substantially affect the functional properties of the resultingprotein molecule are also contemplated. For example, alterations in thegene sequence which reflect the degeneracy of the genetic code, or whichresult in the production of a chemically equivalent amino acid at agiven site, are contemplated; thus, a codon for the amino acid alanine,a hydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine, can also beexpected to produce a biologically equivalent product. Nucleotidechanges which result in alteration of the N-terminal and C-terminalportions of the protein molecule would also not be expected to alter theactivity of the protein. In some cases, it may in fact be desirable tomake mutants of the sequence in order to study the effect of alterationon the biological activity of the protein. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity in the encodedproducts. Moreover, the skilled artisan recognizes that sequencesencompassed by this invention are also defined by their ability tohybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65° C.), withthe sequences exemplified herein.

The term “expression” refers to the transcription and translation togene product from a gene coding for the sequence of the gene product.

The terms “plasmid”, “vector”, and “cassette” as used herein refer to anextra chromosomal element often carrying genes which are not part of thecentral metabolism of the cell and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The terms “transformation” and “transfection” refer to the acquisitionof new genes in a cell after the incorporation of nucleic acid. Theacquired genes may be integrated into chromosomal DNA or introduced asextrachromosomal replicating sequences. The term “transformant” refersto the cell resulting from a transformation.

The term “genetically altered” refers to the process of changinghereditary material by transformation or mutation. The terms“disruption” and “gene interrupt” as applied to genes refer to a methodof genetically altering an organism by adding to or deleting from a genea significant portion of that gene such that the protein encoded by thatgene is either not expressed or not expressed in active form.

Glycerol Biosynthetic Pathway

It is contemplated that glycerol may be produced in recombinantorganisms by the manipulation of the glycerol biosynthetic pathway foundin most microorganisms. Typically, a carbon substrate such as glucose isconverted to glucose-6-phosphate via hexokinase in the presence of ATP.Glucose-phosphate isomerase catalyzes the conversion ofglucose-6-phosphate to fructose-6-phosphate and then tofructose-1,6-diphosphate through the action of 6-phosphofructokinase.The diphosphate is then taken to dihydroxyacetone phosphate (DHAP) viaaldolase. Finally NADH-dependent G3PDH converts DHAP toglycerol-3-phosphate which is then dephosphorylated to glycerol by G3Pphosphatase. (Agarwal (1990), Adv. Biochem. Engrg. 41:114).

Genes Encoding G3PDH, Glycerol Dehydrogenase, G3P Phosphatase andGlycerol Kinase

The present invention provides genes suitable for the expression ofG3PDH and G3P phosphatase activities in a host cell.

Genes encoding G3PDH are known. For example, GPD1 has been isolated fromSaccharomyces and has the base sequence given by SEQ ID NO: 1, encodingthe amino acid sequence given in SEQ ID NO: 7 (Wang et al., supra).Similarly, G3PDH activity has also been isolated from Saccharomycesencoded by GPD2 having the base sequence given in SEQ ID NO: 2 encodingthe amino acid sequence given in SEQ ID NO: 8 (Eriksson et al., (1995)Mol. Microbiol., 17:95).

For the purposes of the present invention it is contemplated that anygene encoding a polypeptide responsible for G3PDH activity is suitablewherein that activity is capable of catalyzing the conversion ofdihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P).Further, it is contemplated that any gene encoding the amino acidsequence of G3PDH as given by SEQ ID NOS: 7, 8, 9, 10, 11 and 12corresponding to the genes GPD1, GPD2, GUT2, gpsA, glpD, and the αsubunit of glpABC respectively, will be functional in the presentinvention wherein that amino acid sequence may encompass amino acidsubstitutions, deletions or additions that do not alter the function ofthe enzyme. The skilled person will appreciate that genes encoding G3PDHisolated from other sources will also be suitable for use in the presentinvention. For example, genes isolated from prokaryotes include GenBankaccessions M34393, M20938, L06231, U12567, L45246, L45323, L45324,L45325, U32164, U32689, and U39682. Genes isolated from fungi includeGenBank accessions U30625, U30876 and X56162; genes isolated frominsects include GenBank accessions X61223 and X14179; and genes isolatedfrom mammalian sources include GenBank accessions U12424, M25558 andX78593.

Genes encoding G3P phosphatase are known. For example, GPP2 has beenisolated from Saccharomyces cerevisiae and has the base sequence givenby SEQ ID NO: 5, which encodes the amino acid sequence given in SEQ IDNO: 13 (Norbeck et al., (1996), J. Biol. Chem., 271:13875).

For the purposes of the present invention, any gene encoding a G3Pphosphatase activity is suitable for use in the method wherein thatactivity is capable of catalyzing the conversion of glycerol-3-phosphateand water to glycerol and inorganic phosphate. Further, any geneencoding the amino acid sequence of G3P phosphatase as given by SEQ IDNOS: 13 and 14 corresponding to the genes GPP2 and GPP1 respectively,will be functional in the present invention including any amino acidsequence that encompasses amino acid substitutions, deletions oradditions that do not alter the function of the G3P phosphatase enzyme.The skilled person will appreciate that genes encoding G3P phosphataseisolated from other sources will also be suitable for use in the presentinvention. For example, the dephosphorylation of glycerol-3-phosphate toyield glycerol may be achieved with one or more of the following generalor specific phosphatases: alkaline phosphatase (EC 3.1.3.1) [GenBankM19159, M29663, U02550 or M33965]; acid phosphatase (EC 3.1.3.2)[GenBank U51210, U19789, U28658 or L20566]; glycerol-3-phosphatase (EC3.1.3.21) [GenBank Z38060 or U18813x11]; glucose-1-phosphatase (EC3.1.3.10) [GenBank M33807]; glucose-6-phosphatase (EC 3.1.3.9) [GenBankU00445]; fructose-1,6-bisphosphatase (EC 3.1.3.11) [GenBank XI 2545 orJ03207] or phosphotidyl glycero phosphate phosphatase (EC 3.1.3.27)[GenBank M23546 and M23628].

Genes encoding glycerol kinase are known. For example, GUT1 encoding theglycerol kinase from Saccharomyces has been isolated and sequenced(Pavlik et al. (1993), Curr. Genet., 24:21) and the base sequence isgiven by SEQ ID NO: 6, which encodes the amino acid sequence given inSEQ ID NO: 15. Alternatively, glpK encodes a glycerol kinase from E.coli and is characterized by the base sequence given in GeneBank L19201,base pairs 77347-78855.

Genes encoding glycerol dehydrogenase are known. For example, gldAencodes a glycerol dehydrogenase from E. coli and is characterized bythe base sequence given in GeneBank U00006, base pairs 3174-4316.Alternatively, dhaD refers to another gene that encodes a glyceroldehydrogenase from Citrobacter freundii and is characterized by the basesequence given in GeneBank U09771, base pairs 2557-3654.

Host Cells

Suitable host cells for the recombinant production of glycerol by theexpression of G3PDH and G3P phosphatase may be either prokaryotic oreukaryotic and will be limited only by their ability to express activeenzymes. Preferred host cells will be those bacteria, yeasts, andfilamentous fungi typically useful for the production of glycerol suchas Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter,Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces,Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula,Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella,Bacillus, Streptomyces and Pseudomonas. Preferred in the presentinvention are E. coli and Saccharomyces.

Where glycerol is a key intermediate in the production of1,3-propane-diol the host cell will either have an endogenous geneencoding a protein having a dehydratase activity or will acquire such agene through transformation. Host cells particularly suited forproduction of 1,3-propanediol are Citrobacter, 796 Enterobacter,Clostridium, Klebsiella, Aerobacter, Lactobacillus, and Salmonella,which have endogenous genes encoding dehydratase enzymes. Additionally,host cells that lack such an endogeneous gene include E. coli.

Vectors and Expression Cassettes

The present invention provides a variety of vectors and transformationand expression cassettes suitable for the cloning, transformation andexpression of G3PDH and G3P phosphatase into a suitable host cell.Suitable vectors will be those which are compatible with the bacteriumemployed. Suitable vectors can be derived, for example, from a bacteria,a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, ayeast or a plant. Protocols for obtaining and using such vectors areknown to those in the art (Sambrook et al., Molecular Cloning: ALaboratory Manual—volumes 1, 2, 3 (Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y., 1989)).

Typically, the vector or cassette contains sequences directingtranscription and translation of the appropriate gene, a selectablemarker, and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. It is mostpreferred when both control regions are derived from genes homologous tothe transformed host cell. Such control regions need not be derived fromthe genes native to the specific species chosen as a production host.

Initiation control regions, or promoters, which are useful to driveexpression of the G3PDH and G3P phosphatase genes in the desired hostcell are numerous and familiar to those skilled in the art. Virtuallyany promoter capable of driving these genes is suitable for the presentinvention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1,PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, and TPI (useful forexpression in Saccharomyces); AOX1 (useful for expression in Pichia);and lac, trp, λP_(L), λP_(R), T7, tac, and trc, (useful for expressionin E. coli).

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary; however, it is most preferred if included.

For effective expression of the instant enzymes, DNA encoding theenzymes are linked operably through initiation codons to selectedexpression control regions such that expression results in the formationof the appropriate messenger RNA.

Transformation of Suitable Hosts and Expression of G3PDH and G3PPhosphatase for the Production of Glycerol

Once suitable cassettes are constructed they are used to transformappropriate host cells. Introduction of the cassette containing thegenes encoding G3PDH and/or G3P phosphatase into the host cell may beaccomplished by known procedures such as by transformation, e.g., usingcalcium-permeabilized cells, electroporation, or by transfection using arecombinant phage virus (Sambrook et al., supra).

In the present invention AH21 and DAR1 cassettes were used to transformthe E. coli DH5α and FM5 as fully described in the GENERAL METHODS andEXAMPLES.

Random and Site Specific Mutagenisis for Disrupting Enzyme Activities:

Enzyme pathways by which organisms metabolize glycerol are known in theart, FIG. 1. Glycerol is converted to glycerol-3-phosphate (G3P) by anATP-dependent glycerol kinase; the G3P may then be oxidized to DHAP byG3PDH. In a second pathway, glycerol is oxidized to dihydroxyacetone(DHA) by a glycerol dehydrogenase; the DHA may then be converted to DHAPby an ATP-dependent DHA kinase. In a third pathway, glycerol is oxidizedto glyceraldehyde by a glycerol dehydrogenase; the glyceraldehyde may bephosphorylated to glyceraldehyde-3-phosphate by an ATP-dependent kinase.DHAP and glyceraldehyde-3-phosphate, interconverted by the action oftriosephosphate isomerase, may be further metabolized via centralmetabolism pathways. These pathways, by introducing by-products, aredeleterious to glycerol production.

One aspect of the present invention is the ability to provide aproduction organism for the production of glycerol where theglycerol-converting activities of glycerol kinase and glyceroldehydrogenase have been deleted. Methods of creating deletion mutantsare common and well known in the art. For example, wild type cells maybe exposed to a variety of agents such as radiation or chemical mutagensand then screened for the desired phenotype. When creating mutationsthrough radiation either ultraviolet (UV) or ionizing radiation may beused. Suitable short wave UV wavelengths for genetic mutations will fallwithin the range of 200 nm to 300 nm where 254 nm is preferred. UVradiation in this wavelength principally causes changes within nucleicacid sequence from guanidine and cytosine to adenine and thymidine.Since all cells have DNA repair mechanisms that would repair most UVinduced mutations, agents such as caffeine and other inhibitors may beadded to interrupt the repair process and maximize the number ofeffective mutations. Long wave UV mutations using light in the 300 nm to400 nm range are also possible but are generally not as effective as theshort wave UV light unless used in conjunction with various activatorssuch as psoralen dyes that interact with the DNA.

Mutagenesis with chemical agents is also effective for generatingmutants and commonly used substances include chemicals that affectnonreplicating DNA such as HNO₂ and NH₂OH, as well as agents that affectreplicating DNA such as acridine dyes, notable for causing frameshiftmutations. Specific methods for creating mutants using radiation orchemical agents are well documented in the art. See for example ThomasD. Brock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition (1989) Sinauer Associates, Inc., Sunderland, Mass., orDeshpande, Mukund V., Appl. Biochem. Biotechnol., 36, 227, (1992),herein incorporated by reference.

After mutagenesis has occurred, mutants having the desired phenotype maybe selected by a variety of methods. Random screening is most commonwhere the mutagenized cells are selected for the ability to produce thedesired product or intermediate. Alternatively, selective isolation ofmutants can be performed by growing a mutagenized population onselective media where only resistant colonies can develop. Methods ofmutant selection are highly developed and well known in the art ofindustrial microbiology. See Brock, Supra., DeMancilha et al., FoodChem., 14, 313, (1984).

Biological mutagenic agents which target genes randomly are well knownin the art. See for example De Bruijn and Rossbach in Methods forGeneral and Molecular Bacteriology (1994) American Society forMicrobiology, Washington, D.C. Alternatively, provided that genesequence is known, chromosomal gene disruption with specific deletion orreplacement is achieved by homologous recombination with an appropriateplasmid. See for example Hamilton et al. (1989) J. Bacteriol.171:4617-4622, Balbas et al. (1993) Gene 136: 211-213, Gueldener et al.(1996) Nucleic Acids Res. 24: 2519-2524, and Smith et al. (1996) MethodsMol. Cell. Biol. 5: 270-277.

It is contemplated that any of the above cited methods may be used forthe deletion or inactivation of glycerol kinase and glyceroldehydrogenase activities in the preferred production organism.

Media and Carbon Substrates

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited tomono-saccharides such as glucose and fructose, oligosaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally, the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated.

Glycerol production from single carbon sources (e.g., methanol,formaldehyde or formate) has been reported in methylotrophic yeasts(Yamada et al. (1989), Agric. Biol. Chem., 53(2):541-543) and inbacteria (Hunter et al. (1985), Biochemistry, 24:4148-4155). Theseorganisms can assimilate single carbon compounds, ranging in oxidationstate from methane to formate, and produce glycerol. The pathway ofcarbon assimilation can be through ribulose monophosphate, throughserine, or through xylulose-monophosphate (Gottschalk, BacterialMetabolism, Second Edition, Springer-Verlag: New York (1986)). Theribulose monophosphate pathway involves the condensation of formate withribulose-5-phosphate to form a 6 carbon sugar that becomes fructose andeventually the three carbon product, glyceraldehyde-3-phosphate.Likewise, the serine pathway assimilates the one-carbon compound intothe glycolytic pathway via methylenetetrahydrofolate.

In addition to one and two carbon substrates, methylotrophic organismsare also known to utilize a number of other carbon-containing compoundssuch as methylamine, glucosamine and a variety of amino acids formetabolic activity. For example, methylotrophic yeast are known toutilize the carbon from methylamine to form trehalose or glycerol(Bellion et al. (1993), Microb. Growth C1 Compd., [Int. Symp.], 7th,415-32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher:Intercept, Andover, UK). Similarly, various species of Candida willmetabolize alanine or oleic acid (Sulter et al. (1990), Arch.Microbiol., 153(5):485-9). Hence, the source of carbon utilized in thepresent invention may encompass a wide variety of carbon-containingsubstrates and will only be limited by the choice of organism.

Although all of the above mentioned carbon substrates and mixturesthereof are suitable in the present invention, preferred carbonsubstrates are monosaccharides, oligosaccharides, polysaccharides,single-carbon substrates or mixtures thereof. More preferred are sugarssuch as glucose, fructose, sucrose, maltose, lactose and single carbonsubstrates such as methanol and carbon dioxide. Most preferred as acarbon substrate is glucose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary forglycerol production.

Culture Conditions

Typically cells are grown at 30° C. in appropriate media. Preferredgrowth media are common commercially prepared media such as LuriaBertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast medium (YM)broth. Other defined or synthetic growth media may also be used and theappropriate medium for growth of the particular microorganism will beknown by one skilled in the art of microbiology or fermentation science.The use of agents known to modulate catabolite repression directly orindirectly, e.g., cyclic adenosine 3′:5′-monophosphate, may also beincorporated into the reaction media. Similarly, the use of agents knownto modulate enzymatic activities (e.g., sulfites, bisulfites, andalkalis) that lead to enhancement of glycerol production may be used inconjunction with or as an alternative to genetic manipulations.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0where the range of pH 6.0 to pH 8.0 is preferred for the initialcondition.

Reactions may be performed under aerobic or anaerobic conditions whereanaerobic or microaerobic conditions are preferred.

Identification of G3PDH, Glycerol Dehydrogenase, G3P Phosphatase, andGlycerol Kinase Activities

The levels of expression of the proteins G3PDH, G3P phosphatase glyceroldehydrogenase, and glycerol kinase are measured by enzyme assays.Generally, G3PDH activity and glycerol dehydrogenase activity assaysrely on the spectral properties of the cosubstrate, NADH, in the DHAPconversion to G-3-P and the DHA conversion to glycerol, respectively.NADH has intrinsic UV/vis absorption and its consumption can bemonitored spectrophotometrically at 340 nm. G3P phosphatase activity canbe measured by any method of measuring the inorganic phosphate liberatedin the reaction. The most commonly used detection method uses thevisible spectroscopic determination of a blue-colored phosphomolybdateammonium complex. Glycerol kinase activity can be measured by thedetection of G3P from glycerol and ATP, for example, by NMR. Assays canbe directed toward more specific characteristics of individual enzymesif necessary, for example, by the use of alternate cofactors.

Identification and Recovery of Glycerol and Other Products (e.g.1,3-propanediol)

Glycerol and other products (e.g. 1,3-propanediol) may be identified andquantified by high performance liquid chromatography (HPLC) and gaschromatography/mass spectroscopy (GC/MS) analyses on the cell-freeextracts. Preferred is a HPLC method where the fermentation media areanalyzed on an analytical ion exchange column using a mobile phase of0.01N sulfuric acid in an isocratic fashion.

Methods for the recovery of glycerol from fermentation media are knownin the art. For example, glycerol can be obtained from cell media bysubjecting the reaction mixture to the following sequence of steps:filtration; water removal; organic solvent extraction; and fractionaldistillation (U.S. Pat. No. 2,986,495).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Production of Glycerol

The present invention describes a method for the production of glycerolfrom a suitable carbon source utilizing a recombinant organism.Particularly suitable in the invention is a bacterial host cell,transformed with an expression cassette carrying either or both of agene that encodes a protein having a glycerol-3-phosphate dehydrogenaseactivity and a gene encoding a protein having a glycerol-3-phosphataseactivity. In addition to the G3PDH and G3P phosphatase genes, the hostcell will contain disruptions in either or both of genes encodingendogenous glycerol kinase and glycerol dehydrogenase enzymes. Thecombined effect of the foreign G3PDH and G3P phosphatase genes(providing a pathway from the carbon source to glycerol) with the genedisruptions (blocking the conversion of glycerol) results in an organismthat is capable of efficient and reliable glycerol production.

Although the optimal organism for glycerol production contains the abovementioned gene disruptions, glycerol production is possible with a hostcell containing either one or both of the foreign G3PDH and G3Pphosphatase genes in the absence of such disruptions. For example, therecombinant E. coli strain AA200 carrying the DAR1 gene (Example 1) wascapable of producing between 0.38 g/L and 0.48 g/L of glycerol dependingon fermentation parameters. Similarly, the E. coli DH5α, carrying andexpressible GPP2 gene (Example 2), was capable of 0.2 g/L of glycerolproduction. Where both genes are present, (Example 3 and 4), glycerolproduction attained about 40 g/L. Where both genes are present inconjunction with an elimination of the endogenous glycerol kinaseactivity, a reduction in the conversion of glycerol may be seen (Example8). Furthermore, the presence of glycerol dehydrogenase activity islinked to the conversion of glycerol under glucose-limited conditions;thus, it is anticipated that the elimination of glycerol dehydrogenaseactivity will result in the reduction of glycerol conversion (Example8).

Production of 1.3-propanediol

The present invention may also be adapted for the production of1,3-propanediol by utilizing recombinant organisms expressing theforeign G3PDH and/or G3P phosphatase genes and containing disruptions inthe endogenous glycerol kinase and/or glycerol dehydrogenase activities.Additionally, the invention provides for the process for the productionof 1,3-propanediol from a recombinant organism where multiple copies ofendogeneous genes are introduced. In addition to these geneticalterations, the production cell will require the presence of a geneencoding an active dehydratase enzyme. The dehydratase enzyme activitymay either be a glycerol dehydratase or a diol dehydratase. Thedehydratase enzyme activity may result from either the expression of anendogenous gene or from the expression of a foreign gene transfectedinto the host organism. Isolation and expression of genes encodingsuitable dehydratase enzymes are well known in the art and are taught byapplicants in PCT/US96/06705, filed 5 Nov. 1996 and U.S. Pat. No.5,686,276 and U.S. Pat. No. 5,633,362, hereby incorporated by reference.It will be appreciated that, as glycerol is a key intermediate in theproduction of 1,3-propanediol, where the host cell contains adehydratase activity in conjunction with expressed foreign G3PDH and/orG3P phosphatase genes and in the absence of the glycerol-convertingglycerol kinase or glycerol dehydrogenase activities, the cell will beparticularly suited for the production of 1,3-propanediol.

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

EXAMPLES

General Methods

Procedures for phosphorylations, ligations, and transformations are wellknown in the art. Techniques suitable for use in the following examplesmay be found in Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press (1989).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found in Manual of Methods forGeneral Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhillips, eds), American Society for Microbiology, Washington, D.C.(1994) or in Biotechnology: A Textbook of Industrial Microbiology(Thomas D. Brock, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass.). All reagents and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

The meaning of abbreviations is as follows: “h” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “1 nL” meansmilliliters, “L” means liters.

Cell Strains

The following Escherichia coli strains were used for transformation andexpression of G3PDH and G3P phosphatase. Strains were obtained from theE. coli Genetic Stock Center, ATCC, or Life Technologies (Gaithersburg,Md.).

-   AA200 (garB10 fhuA22 ompF627 fadL701 relA1 pit-10 spoT1 tpi-1    phoM510 mcrB1) (Anderson et al., (1970), J. Gen. Microbiol.,    62:329).-   BB20 (tonA22 ΔphoA8 fadL701 relA1 glpR2 glpD3 pit-10 gpsA20 spot1    T2R) (Cronan et al., J. Bact., 118:598).-   DH5α (deoR endA1 gyrA96 hsdR17 recA1 relA1 supE44 thi-1    Δ(lacZYA-argFV169) phi80lacZΔM15 F⁻) (Woodcock et al., (1989), Nucl.    Acids Res., 17:3469).-   FM5 Escherichia coli (ATCC 53911)    Identification of Glycerol

The conversion of glucose to glycerol was monitored by HPLC and/or GC.Analyses were performed using standard techniques and materialsavailable to one of skill in the art of chromatography. One suitablemethod utilized a Waters Maxima 820 HPLC system using UV (210 nm) and R1detection. Samples were injected onto a Shodex SH-1011 column (8 mm×300mm; Waters, Milford, Mass.) equipped with a Shodex SH-1011P precolumn (6mm×50 mm), temperature-controlled at 50° C., using 0.01 N H₂SO₄ asmobile phase at a flow rate of 0.69 mL/min. When quantitative analysiswas desired, samples were prepared with a known amount oftrimethylacetic acid as an external standard. Typically, the retentiontimes of 1,3-propanediol (R1 detection), glycerol (R1 detection) andglucose (R1 detection) were 21.39 min, 17.03 min and 12.66 min,respectively.

Glycerol was also analyzed by GC/MS. Gas chromatography with massspectrometry detection for separation and quantitation of glycerol wasperformed using a DB-WAX column (30 m, 0.32 mm I.D., 0.25 um filmthickness, J & W Scientific, Folsom, Calif.) at the followingconditions: injector: split, 1:15; sample volume: 1 uL; temperatureprofile: 150° C. intitial temperature with 30 sec hold, 40° C./min to180° C., 20° C./min to 240° C., hold for 2.5 min. Detection: EI MassSpectrometry (Hewlett Packard 5971, San Fernando, Calif.), quantitativeSIM using ions 61 m/z and 64 m/z as target ions for glycerol andglycerol-d8, and ion 43 m/z as qualifier ion for glycerol. Glycerol-d8was used as an internal standard.

Assay for Glycerol-3-phosphatase, G3P Phosphatase

The assay for enzyme activity was performed by incubating the extractwith an organic phosphate substrate in a bis-Tris or MES and magnesiumbuffer, pH 6.5. The substrate used was either l-α-glycerol phosphate, ord,l-α-glycerol phosphate. The final concentrations of the reagents inthe assay are: buffer (20 mM bis-Tris or 50 mM MES); MgCl₂ (10 mM); andsubstrate (20 mM). If the total protein in the sample was low and novisible precipitation occurs with an acid quench, the sample wasconveniently assayed in the cuvette. This method involved incubating anenzyme sample in a cuvette that contained 20 mM substrate (50 μL, 200mM), 50 mM MES, 10 mM MgCl₂, pH 6.5 buffer. The final phosphatase assayvolume was 0.5 mL. The enzyme-containing sample was added to thereaction mixture; the contents of the cuvette were mixed and then thecuvette was placed in a circulating water bath at T=37° C. for 5 to 120min, the length of time depending on whether the phosphatase activity inthe enzyme sample ranged from 2 to 0.02 U/mL. The enzymatic reaction wasquenched by the addition of the acid molybdate reagent (0.4 mL). Afterthe Fiske SubbaRow reagent (0.1 mL) and distilled water (1.5 mL) wereadded, the solution was mixed and allowed to develop. After 10 min, toallow full color development, the absorbance of the samples was read at660 nm using a Cary 219 UV/Vis spectrophotometer. The amount ofinorganic phosphate released was compared to a standard curve that wasprepared by using a stock inorganic phosphate solution (0.65 mM) andpreparing 6 standards with final inorganic phosphate concentrationsranging from 0.026 to 0.130 μmol/mL.

Spectrophotometric Assay for Glycerol 3-Phosphate Dehydrogenase (G3PDH)Activity

The following procedure was used as modified below from a methodpublished by Bell et al. (1975), J. Biol. Chem., 250:7153-8. This methodinvolved incubating an enzyme sample in a cuvette that contained 0.2 mMNADH; 2.0 mM dihydroxyacetone phosphate (DHAP), and enzyme in 0.1 MTris/HCl, pH 7.5 buffer with 5 mM DTT, in a total volume of 1.0 mL at30° C. The spectrophotometer was set to monitor absorbance changes atthe fixed wavelength of 340 nm. The instrument was blanked on a cuvettecontaining buffer only. After the enzyme was added to the cuvette, anabsorbance reading was taken. The first substrate, NADH (50 uL 4 mMNADH; absorbance should increase approx 1.25 AU), was added to determinethe background rate. The rate should be followed for at least 3 min. Thesecond substrate, DHAP (50 μL 40 mM DHAP), was then added and theabsorbance change over time was monitored for at least 3 min todetermine to determine the gross rate. G3PDH activity was defined bysubtracting the background rate from the gross rate. ₁₃C-NMR Assay forGlycerol Kinase Activity

An appropriate amount of enzyme, typically a cell-free crude extract,was added to a reaction mixture containing 40 mM ATP, 20 mM MgSO₄, 21 mMuniformly 1³C labelled glycerol (99%, Cambridge Isotope Laboratories),and 0.1 M Tris-HCl, pH 9 for 75 min at 25° C. The conversion of glycerolto glycerol 3-phosphate was detected by ¹³C-NMR (125 MHz): glycerol(63.11 ppm, d, J=41 Hz and 72.66 ppm, t, J=41 Hz); glycerol 3-phosphate(62.93 ppm, d, J=41 Hz; 65.31 ppm, br d, J=43 Hz; and 72.66 ppm, dt,J=6, 41 Hz).

NADH-Linked Glycerol Dehydrogenase Assay

NADH-linked glycerol dehydrogenase activity in E. coli strains (gldA)was determined after protein separation by non-denaturing polyacrylamidegel electrophoresis. The conversion of glycerol plus NAD⁺ todihydroxyacetone plus NADH was coupled with the conversion of3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to adeeply colored formazan, using 140:182).

Electrophoresis was performed in duplicate by standard procedures usingnative gels (8-16% TG, 1.5 mm, 15 lane gels from Novex, San Diego,Calif.). Residual glycerol was removed from the gels by washing 3× with50 mM Tris or potassium carbonate buffer, pH 9 for 10 min. The duplicategels were developed, with and without glycerol (approx. 0.16 M finalconcentration), in 15 mL of assay solution containing 50 mM Tris orpotassium carbonate, pH 9, 60 mg ammonium sulfate, 75 mg NAD⁺, 1.5 mgMTT, and 0.5 mg PMS.

The presence or absence of NADH-linked glycerol dehydrogenase activityin E. coli strains (gldA) was also determined, following polyacrylamidegel electrophoresis, by reaction with polyclonal antibodies raised topurified K. pneumoniae glycerol dehydrogenase (dhaD).

Plasmid Construction and Strain Construction

Cloning and Expression of Glycerol 3-Phosphatase for Increase ofGlycerol Production in E. coli DH5α and FM5

The Saccharomyces cerevisiae chromosomeV lamda clone 6592 (Gene Bank,accession # U18813x11) was obtained from ATCC. The glycerol 3-phosphatephosphatase (GPP2) gene was cloned by cloning from the lamda clone astarget DNA using synthetic primers (SEQ ID NO: 16 with SEQ ID NO: 17)incorporating an BamHI-RBS-XbaI site at the 5′ end and a SmaI site atthe 3′ end. The product was subcloned into pCR-Script (Stratagene,Madison, Wis.) at the SrfI site to generate the plasmids pAH15containing GPP2. The plasmid pAH15 contains the GPP2 gene in theinactive orientation for expression from the lac promoter in pCR-ScriptSK+. The BamHI-SmaI fragment from pAH15 containing the GPP2 gene wasinserted into pBlueScriptII SK+ to generate plasmid pAH19. The pAH19contains the GPP2 gene in the correct orientation for expression fromthe lac promoter. The XbaI-PstI fragment from pAH19 containing the GPP2gene was inserted into pPHOX2 to create plasmid pAH21. The pAH21/DH5α isthe expression plasmid.

Plasmids for the Over-Expression of DAR1 in E. coli

DAR1 was Isolated by PCR Cloning from Genomic S. cerevisiae DNA usingsynthetic primers (SEQ ID NO: 18 with SEQ ID NO: 19). Successful PCRcloning places an NcoI site at the 5′ end of DAR1 where the ATG withinNcoI is the DAR1 initiator methionine. At the 3′ end of DAR1 a BamHIsite is introduced following the translation terminator. The PCRfragments were digested with NcoI+BamHI and cloned into the same siteswithin the expression plasmid pTrc99A (Pharmacia, Piscataway, N.J.) togive pDAR1A.

In order to create a better ribosome binding site at the 5′ end of DAR1,an SpeI-RBS-NcoI linker obtained by annealing synthetic primers (SEQ IDNO: 20 with SEQ ID NO: 21) was inserted into the NcoI site of pDAR1A tocreate pAH40. Plasmid pAH40 contains the new RBS and DAR1 gene in thecorrect orientation for expression from the trc promoter of pTrc99A(Pharmacia, Piscataway, N.J.). The NcoI-BamHI fragement from pDAR1A andan second set of SpeI-RBS-NcoI linker obtained by annealing syntheticprimers (SEQ ID NO: 22 with SEQ ID NO: 23) was inserted into theSpeI-BamHI site of pBC-SK+ (Stratagene, Madison, Wis.) to create plasmidpAH42. The plasmid pAH42 contains a chloramphenicol resistant gene.

Construction of Expression Cassettes for DAR1 and GPP2

Expression cassettes for DAR1 and GPP2 were assembled from theindividual DAR1 and GPP2 subclones described above using standardmolecular biology methods. The BamHI-PstI fragment from pAH19 containingthe ribosomal binding site (RBS) and GPP2 gene was inserted into pAH40to create pAH43. The BamHI-PstI fragment from pAH19 containing the RBSand GPP2 gene was inserted into pAH42 to create pAH45.

The ribosome binding site at the 5′ end of GPP2 was modified as follows.A BamHI-RBS-SpeI linker, obtained by annealing synthetic primersGATCCAGGAAACAGA (SEQ ID NO: 24) with CTAGTCTGTTTCCTG (SEQ ID NO: 25) tothe XbaI-PstI fragment from pAH19 containing the GPP2 gene, was insertedinto the BamHI-PstI site of pAH40 to create pAH48. Plasmid pAH48contains the DAR1 gene, the modified RBS, and the GPP2 gene in thecorrect orientation for expression from the trc promoter of pTrc99A(Pharmacia, Piscataway, N.J.).

Transformation of E. coli

All the plasmids described here were transformed into E. coli DH5α orFM5 using standard molecular biology techniques. The transformants wereverified by its DNA RPLP pattern.

Example 1 Production of Glycerol from E. coli Transformed with G3PDHGene

Media

Synthetic media was used for anaerobic or aerobic production of glycerolusing E. coli cells transformed with pDAR1A. The media contained perliter 6.0 g Na₂HPO₄, 3.0 g KH₂PO₄, 1.0 g NH₄Cl, 0.5 g NaCl, 1 mL 20%MgSO₄₀.7H₂O, 8.0 g glucose, 40 mg casamino acids, 0.5 ml 1% thiaminehydrochloride, 100 mg ampicillin.

Growth Conditions

Strain AA200 harboring pDAR1A or the pTrc99A vector was grown in aerobicconditions in 50 mL of media shaking at 250 rpm in 250 mL flasks at 37°C. At A₆₀₀ 0.2-0.3 isopropylthio-β-D-galactoside was added to a finalconcentration of 1 mM and incubation continued for 48 h. For anaerobicgrowth samples of induced cells were used to fill Falcon #2054 tubeswhich were capped and gently mixed by rotation at 37° C. for 48 h.Glycerol production was determined by HPLC analysis of the culturesupernatants. Strain pDAR1A/AA200 produced 0.38 g/L glycerol after 48 hunder anaerobic conditions, and 0.48 g/L under aerobic conditions.

Example 2 Production of Glycerol from E. coli Transformed with G3PPhosphatase Gene (GPP2)

Media

Synthetic phoA media was used in shake flasks to demonstrate theincrease of glycerol by GPP2 expression in E. coli. The phoA mediumcontained per liter: Amisoy, 12 g; ammonium sulfate, 0.62 g; MOPS, 10.5g; Na-citrate, 1.2 g; NaOH (1 M), 10 mL; 1 M MgSO₄, 12 mL; 100× traceelements, 12 mL; 50% glucose, 10 mL; 1% thiamine, 10 mL; 100 mg/mLL-proline, 10 mL; 2.5 mM FeCl₃, 5 mL; mixed phosphates buffer, 2 mL (5mL 0.2 M NaH₂PO₄+9 mL 0.2 M K₂HPO₄), and pH to 7.0. The 100× traceselements for phoA medium/L contained: ZnSO₄.7H₂O, 0.58 g; MnSO₄.H₂O,0.34 g; CuSO₄.5H₂O, 0.49 g; CoCl₂.6H₂O, 0.47 g; H₃BO₃, 0.12 g,NaMoO₄.2H₂O, 0.48 g.

Shake Flasks Experiments

The strains pAH21/DH5α (containing GPP2 gene) and pPHOX2/DH5α (control)were grown in 45 mL of media (phoA media, 50 ug/mL carbenicillin, and 1ug/mL vitamin B₁₂) in a 250 mL shake flask at 37° C. The cultures weregrown under aerobic condition (250 rpm shaking) for 24 h. Glycerolproduction was determined by HPLC analysis of the culture supernatant.pAH21/DH5α produced 0.2 g/L glycerol after 24 h.

Example 3 Production of Glycerol from D-Glucose Using Recombinant E.coli Containing Both GPP2 and DAR1

Growth for demonstration of increased glycerol production by E. coliDH5α-containing pAH43 proceeds aerobically at 37° C. in shake-flaskcultures (erlenmeyer flasks, liquid volume ⅕th of total volume).

Cultures in minimal media/1% glucose shake-flasks are started byinoculation from overnight LB/1% glucose culture with antibioticselection. Minimal media are: filter-sterilized defined media, final pH6.8 (HCl), contained per liter: 12.6 g (NH₄)₂SO₄, 13.7 g K₂HPO₄, 0.2 gyeast extract (Difco), 1 g NaHCO₃, 5 mg vitamin B₁₂, 5 mL ModifiedBalch's Trace-Element Solution (the composition of which can be found inMethods for General and Molecular Bacteriology (P. Gerhardt et al., eds,p. 158, American Society for Microbiology, Washington, D.C. (1994)). Theshake-flasks are incubated at 37° C. with vigorous shaking forovernight, after which they are sampled for GC analysis of thesupernatant. The pAH43/DH5α showed glycerol production of 3.8 g/L after24 h.

Example 4 Production of Glycerol from D-Glucose Using Recombinant E.coli Containing Both GPP2 and DAR1

Example 4 illustrates the production of glucose from the recombinant E.coli DH5α/pAH48, containing both the GPP2 and DAR1 genes.

The strain DH5α/pAH48 was constructed as described above in the GENERALMETHODS.

Pre-Culture

DH5α/pAH48 were pre-cultured for seeding into a fermentation run.Components and protocols for the pre-culture are listed below.Pre-Culture Media KH₂PO₄ 30.0 g/L Citric acid 2.0 g/L MgSO₄.7H₂O 2.0 g/L98% H₂SO₄ 2.0 mL/L Ferric ammonium citrate 0.3 g/L CaCl₂.2H₂O 0.2 g/LYeast extract 5.0 g/L Trace metals 5.0 mL/L Glucose 10.0 g/LCarbenicillin 100.0 mg/L

The above media components were mixed together and the pH adjusted to6.8 with NH₄OH. The media was then filter sterilized.

Trace metals were used according to the following recipe: Citric acid,monohydrate 4.0 g/L MgSO₄.7H₂O 3.0 g/L MnSO4.H₂O 0.5 g/L NaCl 1.0 g/LFeSO4.7H₂O 0.1 g/L CoCl2.6H₂O 0.1 g/L CaCl₂ 0.1 g/L ZnSO₄.7H₂O 0.1 g/LCuSO₄.5 H₂O 10 mg/L AlK(SO₄)₂.12H₂O 10 mg/L H₃BO₃ 10 mg/L Na₂MoO₄.2H₂O10 mg/L NiSO4.6H₂O 10 mg/L Na₂SeO₃ 10 mg/L Na₂WO₄.2H₂O 10 mg/L

Cultures were started from seed culture inoculated from 50 μL frozenstock (15% glycerol as cryoprotectant) to 600 mL medium in a 2-LErlenmeyer flask. Cultures were grown at 30° C. in a shaker at 250 rpmfor approximately 12 h and then used to seed the fermenter. Fermentationgrowth Vessel 15-L stirred tank fermenter Medium KH₂PO₄ 6.8 g/L Citricacid 2.0 g/L MgSO₄.7H₂O 2.0 g/L 98% H₂SO₄ 2.0 mL/L Ferric ammoniumcitrate 0.3 g/L CaCl₂.2H₂O 0.2 g/L Mazu DF204 antifoam 1.0 mL/L

The above components were sterilized together in the fermenter vessel.

The pH was raised to 6.7 with NH₄OH. Yeast extract (5 g/L) and tracemetals solution (5 mL/L) were added aseptically from filter sterilizedstock solutions. Glucose was added from 60% feed to give finalconcentration of 10 g/L. Carbenicillin was added at 100 mg/L. Volumeafter inoculation was 6 L.

Environmental Conditions for Fermentation

The temperature was controlled at 36° C. and the air flow rate wascontrolled at 6 standard liters per minute. Back pressure was controlledat 0.5 bar. The agitator was set at 350 rpm. Aqueous ammonia was used tocontrol pH at 6.7. The glucose feed (60% glucose monohydrate) rate wascontrolled to maintain excess glucose.

Results

The results of the fermentation run are given in Table 1. TABLE 1 EFTOD550 [Glucose] [Glycerol] Total Glucose Total Glycerol (hr) (AU) (g/L)(g/L) Fed (g) Produced (g) 0 0.8 9.3 25 6 4.7 4.0 2.0 49 14 8 5.4 0 3.671 25 10 6.7 0.0 4.7 116 33 12 7.4 2.1 7.0 157 49 14.2 10.4 0.3 10.0 23070 16.2 18.1 9.7 15.5 259 106 18.2 12.4 14.5 305 20.2 11.8 17.4 17.7 353119 22.2 11.0 12.6 382 24.2 10.8 6.5 26.6 404 178 26.2 10.9 6.8 442 28.210.4 10.3 31.5 463 216 30.2 10.2 13.1 30.4 493 213 32.2 10.1 8.1 28.2512 196 34.2 10.2 3.5 33.4 530 223 36.2 10.1 5.8 548 38.2 9.8 5.1 36.1512 233

Example 5 Engineering of Glycerol Kinase Mutants of E. coli FM5 forProduction of Glycerol from Glucose

Construction of Integration Plasmid for Glycerol Kinase Gene Replacementin E. coli FM5

E. coli FM5 genomic DNA was prepared using the Puregene DNA IsolationKit (Gentra Systems, Minreapolis, Minn.). A 1.0 kb DNA fragmentcontaining partial glpF and glycerol kinase (glpK) genes was amplifiedby PCR (Mullis and Faloona, Methods Enzymol., 155:335-350, 1987) fromFM5 genomic DNA using primers SEQ ID NO: 26 and SEQ ID NO: 27. A 1.1 kbDNA fragment containing partial glpK and glpX genes was amplified by PCRfrom FM5 genomic DNA using primers SEQ ID NO: 28 and SEQ ID NO: 29. AMunI site was incorporated into primer SEQ ID NO: 28. The 5′ end ofprimer SEQ ID NO: 28 was the reverse complement of primer SEQ ID NO: 27to enable subsequent overlap extension PCR. The gene splicing by overlapextension technique (Horton et al., BioTechniques, 8:528-535, 1990) wasused to generate a 2.1 kb fragment by PCR using the above two PCRfragments as templates and primers SEQ ID NO: 26 and SEQ ID NO: 29. Thisfragment represented a deletion of 0.8 kb from the central region of the1.5 kb glpK gene. Overall, this fragment had 1.0 kb and 1.1 kb flankingregions on either side of the MunI cloning site (within the partialglpK) to allow for chromosomal gene replacement by homologousrecombination.

The above 2.1 kb PCR fragment was blunt-ended (using mung bean nuclease)and cloned into the pCR-Blunt vector using the Zero Blunt PCR CloningKit (Invitrogen, San Diego, Calif.) to yield the 5.6 kb plasmid PRN100containing kanamycin and Zeocin resistance genes. The 1.2 kb HincIIfragment from pLoxCat1 (unpublished results), containing achloramphenicol-resistance gene flanked by bacteriophage P1 loxP sites(Snaith et al., Gene, 166:173-174, 1995), was used to interrupt the glpKfragment in plasmid pRN100 by ligating it to MunI-digested (andblunt-ended) plasmid pRN100 to yield the 6.9 kb plasmid pRN101-1. A 376bp fragment containing the R6K origin was amplified by PCR from thevector pGP704 (Miller and Mekalanos, J. Bacteriol., 170:2575-2583, 1988)using primers SEQ ID NO: 30 and SEQ ID NO: 31, blunt-ended, and ligatedto the 5.3 kb Asp718-AatII fragment (which was blunt-ended) frompRN101-1 to yield the 5.7 kb plasmid pRN102-1 containing kanamycin andchloramphenicol resistance genes. Substitution of the ColE1 originregion in pRN101-1 with the R6K origin to generate pRN102-1 alsoinvolved deletion of most of the Zeocin resistance gene. The host forpRN102-1 replication was E. coli SY327 (Miller and Mekalanos, J.Bacteriol., 170:2575-2583, 1988) which contains the pir gene necessaryfor the function of the R6K origin.

Engineering of Glycerol Kinase Mutant RJF10m with ChloramphenicolResistance Gene Interrupt

E. coli FM5 was electrotransformed with the non-replicative integrationplasmid pRN102-1 and transformants that were chloramphenicol-resistant(12.5 μg/mL) and kanamycin-sensitive (30 μg/mL) were further screenedfor glycerol non-utilization on M9 minimal medium containing 1 mMglycerol. An EcoRI digest of genomic DNA from one such mutant, RJF10m,when probed with the intact glpK gene via Southern analysis (Southern,J. Mol. Biol., 98:503-517, 1975) indicated that it was adouble-crossover integrant (glpK gene replacement) since the twoexpected 7.9 kb and 2.0 kb bands were observed, owing to the presence ofan additional EcoRI site within the chloramphenicol resistance gene. Thewild-type control yielded the single expected 9.4 kb band. A ¹³C NMRanalysis of mutant RJF10m confirmed that it was incapable of converting¹³C-labeled glycerol and ATP to glycerol-3-phosphate. This glpK mutantwas further analyzed by genomic PCR using primer combinations SEQ ID NO:32 and SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, and SEQ ID NO: 32and SEQ ID NO: 35 which yielded the expected 2.3 kb, 2.4 kb, and 4.0 kbPCR fragments respectively. The wild-type control yielded the expected3.5 kb band with primers SEQ ID NO: 32 and SEQ ID NO: 35. The glpKmutant RJF10m was electrotransformed with plasmid pAH48 to allowglycerol production from glucose. The glpK mutant E. coli RJF10m hasbeen deposited with ATCC under the terms of the Budapest Treaty on 24Nov. 1997.

Engineering of Glycerol Kinase Mutant RJF10 with ChloramphenicolResistance Gene Interrupt Removed

After overnight growth on YENB medium (0.75% yeast extract, 0.8%nutrient broth) at 37° C., E. coli RJF10m in a water suspension waselectrotransformed with plasmid pJW168 (unpublished results), whichcontained the bacteriophage P1 Cre recombinase gene under the control ofthe IPTG-inducible lacUV5 promoter, a temperature-sensitive pSC101replicon, and an ampicillin resistance gene. Upon outgrowth in SOCmedium at 30° C., transformants were selected at 30° C. (permissivetemperature for pJW168 replication) on LB agar medium supplemented withcarbenicillin (50 μg/mL) and IPTG (1 mM). Two serial overnight transfersof pooled colonies were carried out at 30° C. on fresh LB agar mediumsupplemented with carbenicillin and IPTG in order to allow excision ofthe chromosomal chloramphenicol resistance gene via recombination at theloxP sites mediated by the Cre recombinase (Hoess and Abremski, J. Mol.Biol., 181:351-362, 1985). Resultant colonies were replica-plated on toLB agar medium supplemented with carbenicillin and IPTG and LB agarsupplemented with chloramphenicol (12.5 μg/mL) to identify colonies thatwere carbenicillin-resistant and chloramphenicol-sensitive indicatingmarker gene removal. An overnight 30° C. culture of one such colony wasused to inoculate 10 mL of LB medium. Upon growth at 30° C. to OD (600nm) of 0.6, the culture was incubated at 37° C. overnight. Severaldilutions were plated on prewarmed LB agar medium and the platesincubated overnight at 42° C. (the non-permissive temperature for pJW168replication). Resultant colonies were replica-plated on to LB agarmedium and LB agar medium supplemented with carbenicillin (75 μg/mL) toidentify colonies that were carbenicillin-sensitive indicating loss ofplasmid pJW168. One such glpK mutant, RJF10, was further analyzed bygenomic PCR using primers SEQ ID NO: 32 and SEQ ID NO: 35 and yieldedthe expected 3.0 kb band confirming marker gene excision. Glycerolnon-utilization by mutant RJF10 was confirmed by lack of growth on M9minimal medium containing 1 mM glycerol. The glpK mutant RJF10 waselectrotransformed with plasmid pAH48 to allow glycerol production fromglucose.

Example 6 Construction of E. coli Strain with GLDA Gene Knockout

The gldA gene was isolated from E. coli by PCR (K. B. Mullis and F. A.Faloona (1987) Meth. Enzymol. 155:335-350) using primers SEQ ID NO: 36and SEQ ID NO: 37, which incorporate terminal Sph1 and Xba1 sites,respectively, and cloned (T. Maniatis 1982 Molecular Cloning. ALaboratory Manual. Cold Spring Harbor, Cold Spring Harbor, N.Y.) betweenthe Sph1 and Xba1 sites in pUC18, to generate pKP8. pKP8 was cut at theunique Sal1 and Nco1 sites within the gldA gene, the ends flushed withKlenow and religated, resulting in a 109 bp deletion in the middle ofgldA and regeneration of a unique Sal1 site, to generate pKP9. A 1.4 kbDNA fragment containing the gene conferring kanamycin resistance (kan),and including about 400 bps of DNA upstream of the translational startcodon and about 100 bps of DNA downstream of the translational stopcodon, was isolated from pET-28a(+) (Novagen, Madison, Wis.) by PCRusing primers SEQ ID NO: 38 and SEQ ID NO: 39, which incorporateterminal Sal1 sites, and subcloned into the unique Sal1 site of pKP9, togenerate pKP13. A 2.1 kb DNA fragment beginning 204 bps downstream ofthe gldA translational start codon and ending 178 bps upstream of thegldA translational stop codon, and containing the kan insertion, wasisolated from pKP13 by PCR using primers SEQ ID NO: 40 and SEQ ID NO:41, which incorporate terminal Sph1 and Xba1 sites, respectively, wassubcloned between the Sph1 and Xba1 sites in pMAK705 (GenencorInternational, Palo Alto, Calif.), to generate pMP33. E. coli FM5 wastransformed with pMP33 and selected on 20 ug/mL kan at 30° C., which isthe permissive temperature for pMAK705 replication. One colony wasexpanded overnight at 30° C. in liquid media supplemented with 20 ug/mLkan. Approximately 32,000 cells were plated on 20 ug/mL kan andincubated for 16 hrs at 44° C., which is the restrictive temperature forpMAK705 replication. Transformants growing at 44° C. have plasmidintegrated into the chromosome, occuring at a frequency of approximately0.0001. PCR and Southern blot (E.M. Southern 1975 J. Mol. Biol.98:503-517) analyses were used to determine the nature of thechromosomal integration events in the transformants. Western blotanalysis (H. Towbin, et al. (1979) Proc. Natl. Acad. Sci. 76:4350) wasused to determine whether glycerol dehydrogenase protein, the product ofgldA, is produced in the transformants. An activity assay was used todetermine whether glycerol dehydrogenase activity remained in thetransformants. Activity in glycerol dehydrogenase bands on native gelswas determined by coupling the conversion ofglycerol+NAD(+)→dihydroxyacetone+NADH to the conversion of a tetrazoliumdye, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]to a deeply colored formazan, with phenazine methosulfate as mediator.Glycerol dehydrogenase also requires the presence of 30 mM ammoniumsulfate and 100 mM Tris, pH 9 (C.-T. Tang, et al. (1997) J. Bacteriol.140:182). Of 8 transformants analyzed, 6 were determined to be gldAknockouts. E. coli MSP33.6 has been deposited with ATCC under the termsof the Budapest Treaty on 24 Nov. 1997.

Example 7 Construction of E. coli Strain with GLPK and GLDA Geneknockouts

A 1.6 kb DNA fragment containing the gldA gene, and including 228 bps ofDNA upstream of the translational start codon and 220 bps of DNAdownstream of the translational stop codon was isolated from E. coli byPCR using primers SEQ ID NO: 42 and SEQ ID NO: 43, which incorporateterminal Sph1 and Xba1 sites, repectively, and cloned between the Sph1and Xba1 sites of pUC18, to generate pQN2. pQN2 was cut at the uniqueSal1 and Nco1 sites within the gldA gene, the ends flushed with Klenowand religated, resulting in a 109 bp deletion in the middle of gldA andregeneration of a unique Sal1 site, to generate pQN4. A 1.2 kb DNAfragment containing the gene conferring kanamycin resistance (kan), andflanked by loxP sites was isolated from pLoxKan2 (Genecor International,Palo Alto, Calif.) as a Stu1/Xho1 fragment, the ends flushed withKlenow, and subcloned into pQN4 at the Sal1 site after flushing withKlenow, to generate pQN8. The Sph1/Xba1 fragment from pQN8 containingthe kan-interupted gldA was subcloned between the Sph1 and Xba1 sites ofpGP704, using E. coli SY327 as host, to generate pQN9. E. coli RJF10(see EXAMPLE 5) was transformed with pQN9 and selected on kan. Since thepGP704 backbone cannot replicate in most E. coli hosts, transformantsarise by integration of the plasmid, (or portions of it) into thechromosome. Double crossover integration events are determined byidentifying those transformants which are kan resistant and ampicillinsensitive. PCR and Southern blot analyses are used to determine thenature of the chromosomal integration events in the transformants.Western blot analysis is used to determine whether glyceroldehydrogenase, the product of gldA, is produced in the transformants.Activity assays are used to determine whether glycerol dehydrogenaseactivity remains in the transformants. The kan marker is removed fromthe chromosome using the Cre-producing plasmid pJW168, as described inEXAMPLE 5.

Example 8 Consumption of Glycerol Produced from D-Glucose by RecombinantE. coli Containing both GPP2 and DAR1 with and without Glycerol Kinase(GLPK) Activity

EXAMPLE 8 illustrates the consumption of glycerol by the recombinant E.coli FM5/pAH48 and RJF10/pAH48. The strains FM5/pAH48 and RJF10/pAH48were constructed as described above in the GENERAL METHODS.

Pre-Culture

FM5/pAH48 and RJF10/pAH48 were pre-cultured for seeding a fermenter inthe same medium used for fermentation, or in LB supplemented with 1%glucose. Either carbenicillin or ampicillin were used (100 mg/L) forplasmid maintenance. The medium for fermentation is as described inEXAMPLE 4.

Cultures were started from frozen stocks (15% glycerol ascryoprotectant) in 600 mL medium in a 2-L Erlenmeyer flask, grown at 30°C. in a shaker at 250 rpm for approximately 12 h, and used to seed thefermenter.

Fermentation Growth

A 15-L stirred tank fermenter with 5-7 L initial volume was prepared asdescribed in EXAMPLE 4. Either carbenicillin or ampicillin were used(100 mg/L) for plasmid maintenance.

Environmental Conditions to Evaluate Glycerol Kinase (GlpK) Activity

The temperature was controlled at 30° C. and the air flow ratecontrolled at 6 standard liters per minute. Back pressure was controlledat 0.5 bar. Dissolved oxygen tension was controlled at 10% by stirring.Aqueous ammonia was used to control pH at 6.7. The glucose feed (60%glucose) rate was controlled to maintain excess glucose until glycerolhad accumulated to at least 25 g/L. Glucose was then depleted, resultingin the net metabolism of glycerol. Table 2 shows the resultingconversion of glycerol. TABLE 2 Conversion of glycerol by FM5/pAH48 (wt)and RJF10/pAH48 (glpK) rate of glycero consumption Strain number ofexamples g/OD/hr FM5/pAH48 2 0.095 ± 0.015 RJF10/pAH48 3 0.021 ± 0.011

As is seen by the data in Table 2, the rate of glycerol consumptiondecreases about 4-5 fold where endogenous glycerol kinase activity iseliminated.

Environmental Conditions to Evaluate Glycerol Dehydrogenase (GldA)Activity

The temperature was controlled at 30° C. and the air flow ratecontrolled at 6 standard liters per minute. Back pressure was controlledat 0.5 bar. Dissolved oxygen tension was controlled at 10% by stirring.Aqueous ammonia was used to control pH at 6.7. In the firstfermentation, glucose was kept in excess for the duration of thefermentation. The second fermentation was operated with no residualglucose after the first 25 hours. Samples over time from the twofermentations were taken for evaluation of GlpK and GldA activities.Table 3 summarizes RJF10/pAH48 fermentations that show the effects ofGldA on selectivity for glycerol. TABLE 3 GldA and GlpK activititiesfrom two RJF10/pAH48 fermentations Time Overall selectivity Fermentation(hrs) GldA GlpK (g/g) 1 25 − − 42% 46 − − 49% 61 + − 54% 2 25 + − 41% 46++ − 14% 61 ++ − 12%

As is seen by the data in Table 3, the presence of glyceroldehydrogenase (GlDA) activity is linked to the conversion of glycerolunder glucose-limited conditions; thus, it is anticipated thateliminating glycerol dehydrogenase activity will reduce glycerolconversion.

1-16. (canceled)
 17. A method for the production of 1,3-propanediol froma recombinant organism comprising: (i) transforming a suitable host cellwith an expression cassette comprising either one or both of (a) a geneencoding a protein having glycerol-3-phosphate dehydrogenase activity,and (b) a gene encoding a protein having glycerol-3-phosphatephosphatase activity, the suitable host cell having at least one geneencoding a protein having a dehydratase activity and having a disruptionin either one or both of: (a) an endogenous gene encoding a polypeptidehaving glycerol kinase activity, and (b) an endogenous gene encoding apolypeptide having glycerol dehydrogenase activity, wherein thedisruption in the genes of (a) or (b) prevents the expression of activegene product; (ii) culturing the transformed host cell of (i) in thepresence of at least one carbon source selected from the groupconsisting of monosaccharides, oligosaccharides, polysaccharides, andsingle-carbon substrates whereby 1,3-propanediol is produced; and (iii)recovering the 1,3-propanediol produced in (ii).
 18. The method of claim17 wherein the protein having a dehydratase activity is selected fromthe group consisting of a glycerol dehydratase enzyme and a dioldehydratase enzyme.
 19. The method of claim 18 wherein the glyceroldehydratase enzyme is encoded by a gene, the gene isolated from amicroorganism, the microorganism selected from the group consisting ofKlebsiella, Lactobacillus, Enterobacter, Citrobacter, Pelobacter,Ilyobacter, and Clostridium.
 20. The method of claim 18 wherein the dioldehydratase enzyme is encoded by a gene, the gene isolated from amicroorganism, the microorganism selected from the group consisting ofKlebsiella and Salmonella.
 21. (canceled)